We identified three genes homologous to water channels in the plasma membrane type subfamily from roots of barley seedlings. These genes were designated HvPIP2;1, HvPIP1;3, and HvPIP1;5 after comparison to Arabidopsis aquaporins. Competitive reverse transcription (RT)-PCR was applied in order to distinguish and to quantify their transcripts. The HvPIP2;1 transcript was the most abundant among the three in roots. Salt stress (200 mM NaCl) down-regulated HvPIP2;1 (transcript and protein), but had almost no effect on the expressions of HvPIP1;3, or HvPIP1;5. Approximately equal amounts of the transcripts of the three were detected in shoots, and salt stress enhanced the expression of HvPIP2;1 but not of HvPIP1;3, or HvPIP1;5. HvPIP2;1 protein was confirmed to be localized in the plasma membrane. Functional expression of HvPIP2;1 in Xenopus oocytes confirmed that HvPIP2;1 encoded an aquaporin that transports water. This water permeability was reduced by HgCl(2), which is a typical water channel inhibitor. This activity was not modified by some inhibitors against protein kinase and protein phosphatase.

Despite attempts in a number of studies to utilize muscle tissue and cells as microactuators, all of the biohybrid microdevices have been operable only in the culture medium and none have worked in air due to the dry environment. This paper demonstrates an atmospheric-operable bioactuator (AOB) fabricated by packaging an insect dorsal vessel (DV) tissue with a small amount of culture medium inside a capsule. The AOB, consisting of microtweezers and the capsule, was designed based on a structural simulation that took into account the capillary effect. The base part of the microtweezers was deformed by spontaneous contractions of the DV tissue in the medium inside the capsule, by which the front edges of the microtweezer arms projecting above the medium surface were also deformed. First, we confirmed in the medium that the DV tissue was able to reduce the gap between the arm tips of the microtweezers. After taking the AOB out of the medium, as we expected, the AOB continued to work in air at room temperature. The gap reduction in air became larger than the one in the medium due to a surface tension effect, which was consistent with the simulation findings on the surface tension by the phase-field method. Second, we demonstrated that the AOB deformed a thin-wall ring placed between its tips in air. Third, we measured the lifetime of the AOB. The AOB kept working for around 40 minutes in air, but eventually stopped due to medium evaporation. As the evaporation progressed, the microtweezers were pressed onto the capsule wall by the surface tension and opening and closing stopped. Finally, we attempted to prevent the medium from evaporating by pouring liquid paraffin (l-paraffin) over the medium after lipophilic coating of the capsule. As a result, we succeeded in prolonging the AOB lifetime to more than five days. In this study, we demonstrated the significant potential of insect muscle tissue and cells as a bioactuator in air and at room temperature. By integrating insect tissue and cells not only into a microspace but also onto a substrate, we expect to realize a biohybrid MEMS device with various functions in the near future.

We present a bioactuator powered by insect dorsal vessel tissue which can work for a long time at room temperature without maintenance. Previously reported bioactuators which exploit contracting ability of mammalian heart muscle cell have required precise environmental control to keep the cell alive and contracting. To overcome this problem, we propose a bioactuator using dorsal vessel tissue. The insect tissue which can grow at room temperature is generally robust over a range of culture conditions compared to mammalian tissues and cells. First, we confirm that a dorsal vessel tissue of lepidoptera larva Ctenoplusia agnata contracts spontaneously for at least 30 days without medium replacement at 25 degrees C. Using the dorsal vessel tissue cultured under the same conditions, we succeed in driving micropillars 100 microm in diameter and 1000 microm in height for more than 90 days. The strongest displacement of the micropillar top occurs on the 42(nd) day and is 23 microm. Based on these results, the contracting force is roughly estimated as 4.7 microN which is larger than that by a few mammalian cardiomyocytes (3.4 microN). Definite displacements of more than 10 microm are observed for 58 days from the 15(th) to the 72(nd) days. The number of life cycles can be roughly calculated as 7.5 x 10(5) times for the average frequency of about 0.15 Hz, which is no less than that of conventional mechanical actuators. These results suggest that the insect dorsal vessel tissue is a more promising material for bioactuators used at room temperature than other biological cell-based materials.

Cryopreservation is widely used to maintain backups of cells as it enables the semipermanent storage of cells. During the freezing process, ice crystals that are generated inside and outside the cells can lethally damage the cells. All conventional cryopreservation methods use at least one cryoprotective agent (CPA) to render water inside and outside the cells vitreous or nanocrystallized (nearvitrification) without forming damaging ice crystals. However, CPAs should ideally be avoided due to their cytotoxicity and potential side effects on the cellular state. Herein, we demonstrate the CPAfree cryopreservation of mammalian cells by ultrarapid cooling using inkjet cell printing, which we named superflash freezing (SFF). The SFF cooling rate, which was estimated by a heat-transfer stimulation, is sufficient to nearly vitrify the cells. The experimental results of Raman spectroscopy measurements, and observations with an ultrahigh-speed video camera support the near-vitrification of the droplets under these conditions. Initially, the practical utility of SFF was demonstrated on mouse fibroblast 3T3 cells, and the results were comparable to conventional CPA-assisted methods. Then, the general viability of this method was confirmed on mouse myoblast C2C12 cells and rat primary mesenchymal stem cells. In their entirety, the thus-obtained results unequivocally demonstrate that CPA-free cell cryopreservation is possible by SFF. Such a CPA-free cryopreservation method should be ideally suited for most cells and circumvent the problems typically associated with the addition of CPAs. cryopreservation | superflash freezing | cryoprotectant agent-free | inkjet cell printing | vitrification

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